Method for screening of modulators of calcineurin activity

ABSTRACT

A method for screening of modulators of calcineurin is provided, which uses the interaction between calcineurin and superoxide dismutase. Modulators of calcineurin are potential candidates for drugs, e.g. for immunosuppressive drugs. The forming of a complex comprising calcineurin and superoxide dismutase is monitorable in the presence of potential activators or inhibitors of calcineurin. Complex formation is performed within the cell by the use of appropriate expression vectors or in vitro using isolated proteins. Preferably, complex formation is monitored by fluorescence detection, especially by laser fluctuation correlation spectroscopy.

This application claims the benefit to foreign priority under 35 U.S.C.§119 of European Patent Application No. 98113876.1, filed on Jul. 22,1998, the contents of which are hereby incorporated by reference intheir entirety.

The invention relates to a method for screening of modulators ofcalcineurin.

Calcineurin (E.C. 3.1.3.16) is a serine/threonine phospho-proteinphosphatase and is composed of a catalytic (calcineurin A) andregulatory (calcineurin B) subunit (about 60 and about 18 kDa,respectively). In mammals, three distinct genes (A-alpha, A-beta,A-gamma) for the catalytic subunit have been characterized, each ofwhich can undergo alternative splicing to yield additional variants.Although mRNA for all three genes appears to be expressed in mosttissues, two isoforms (A-alpha and A-beta) are most predominant inbrain.

Calcineurin has been cloned from various organisms including human(Guerini et al., 1989), (Guerini and Klee, 1989), (Kincaid et al.,1991), (Kuno et al., 1989), (Ito et al., 1989), (Muramatsu and Kincaid,1993). The crystal structure has shown that calcineurin A contains abinuclear metal center with unknown enzymatic function (Griffith et al.,1995). Recombinant expression of rat calcineurin A subunit in bacteriaor SF9-cells were not effective and yielded only poor enzymaticactivities since calcineurin A is not stable in the absence ofcalcineurin B (Perrino et al., 1992), (Perrino et al., 1995), (Haddy andRusnack, 1994). Coexpression of calcineurin A and calcium bindingsubunit calcineurin B yielded a more stable and active enzyme (Mondragonet al., 1997). Calcineurin has been implicated in various neuronalsignaling pathways (Klee et al., 1988), (Yakel, 1997) but the neuronalfunction is only poorly understood (Guerini, 1997).

Calcineurin is the only protein phosphatase known to be under thecontrol of Ca²⁺ and calmodulin. Binding of Ca²⁺ and calmodulin isnecessary for enzymatic activity. Calmodulin is bound by the catalyticsubunit whereas the regulatory subunit possesses four Ca²⁺ bindingsites.

Calcineurin is discussed in the context of immunosuppression. It hasbeen shown that calcineurin acts via the transcription factor NFAT(nuclear factor of activated T cells) on the T cell response. Thefunctions of NFAT proteins are directly controlled by calcineurin in acalcium- and calmodulin-dependent manner. Activation of NFAT bycalcineurin is mediated by the cytosolic binding protein FKBP.

Substances which are able to block the calcineurin signal pathway aresuitable agents in order to block the T cell activation and therebysuppressing the immune response. Suppression of immune response hasimportant clinical relevance, for example in transplantation surgery forpreventing rejection episodes. Therefore, calcineurin as pharmacologicaltarget is of great importance and several attemps were made to developagents which block the calcineurin signal pathway. Examples of suchimmunosuppressive drugs are FK506 (Fujisawa) and cyclosporine (Novartis)(Liu et al, 1991). These antibiotics inhibit calcineurin phosphataseactivity in the presence of immunophilin receptor proteins (FKBP,cyclophillin) and thereby suppress immune response by preventing theactivation of the T cell transcription factor NFAT (Liu et al., 1992),(Nelson et al., 1993). FK50G (tacrolimus) binds to the binding proteinFKBP and thereby prevents calcineurin from binding to FKBP. Accordinglythe signal pathway is interrupted. No activation of the transcriptionfactor NFAT is achieved and the T cell activation is disturbed.

Nevertheless, there are several severe disadvantages and side-effect ofsaid drugs. In clinical trials with liver and renal transplantrecipients it has been shown that FK506-based therapy was associatedwith increased toxicities in comparison to conventional therapy.Furthermore FK506 has negative effects on the bone mineral physiology.

Besides the role of the calcineurin signal pathway in immune response ithas been shown that calcineurin is involved in apoptosis induction byglutamate excitotoxicity in neuronal cells (Ankarcrona et al., 1996) Lowenzymatic levels of calcineurin have been associated with Alzheimersdisease (Ladner et al., 1996), (Kayyali et al., 1997). Calcineurininhibitors (FK506, Cyclosporin) prevented epileptogenesis in modelorganisms (Moriwaki et al., 1996). In the heart or in the braincalcineurin also plays a key role in the stress response after hypoxiaor ischemia (Butcher et al., 1997), (Hashimoto et al., 1998), (Molkentinet al., 1998).

In summary, calcineurin is a crucial target to develop new substancessuitable as drugs, especially as immunosuppressive drugs. Formerscreening systems using purified calcineurin and conventional assayslike radioactive or HPLC assays (Klee, 1991), (Enz et al., 1994) did notlead to appropriate new substances. Therefore, the invention has theobject to provide a new screening system for modulators of calcineurintaking advantage of new insights into the signal pathway of calcineurin.By the use of this new screening system it is possible to develop newpharmaceuticals with respect to the field of transplantation surgery,cardiac infarction and apoplexy, chronic or acute neurodegeneration andinflammatory diseases, for example. This object is solved by a method asdescribed herein. Preferred embodiments of the inventive method arefurther described herein. A kit, vectors, cells and a peptide suitablefor performing the inventive method are also described herein. Thewording of all claims is hereby incorporated in the specification byreference.

The inventive method is based on results showing that a physiologicalinteraction between calcineurin and superoxide dismutase takes placewhich provides a suitable target for developing of a new screeningsystem.

For a long time it was not understood why recombinant or even purifiedcalcineurin exhibited only 1 to 2% of the specific activity estimated incrude brain extracts until it was detected that the binuclear metalcenter of the enzyme contains a redoxsensitive Fe²⁺ (Yu et al., 1997).After calcium activation or during purification procedure the Fe²⁺ isoxidized by oxygen species and turns the enzyme inactive (Stemmer etal., 1995), (Wang et al., 1996).

Recently it has been shown that copper/zinc superoxide dismutase(CuZnSOD, EC 1.15.1.1) protects calcineurin against oxidativeinactivation (Wang et al., 1996). The phosphatase activity ofcalcineurin is strongly dependent on the presence of calcium andcalmodulin. The addition of Ca²⁺ in the presence of calmodulin leads toa drastic increase in activity. But during several minutes this activityis lost. By the addition of copper/zinc superoxide dismutase theactivity can be maintained.

Superoxide dismutase (SOD) dismutates the hyperoxide anion (superoxide)into hydroperoxide and molecular oxygen. There are two forms of thisenzyme: the mitochondrial form containing manganese and the cytosolicform containing copper and zinc. In general superoxide dismutase isconsidered to be a catcher of radicals and is discussed in the field ofdetoxification of reactive oxygen species. Therefore, the role ofsuperoxide dismutase in the protection of the activity of calcineurinfound by Wang et al. was considered to be the result of general redoxfunction of superoxide dismutase. Now, surprising results of theinventor lead to the knowledge that a physiological interaction betweencalcineurin and superoxide dismutase takes place. Several mutants ofcopper/zinc superoxide dismutase lacking the enzymatic function showedthe protective effect on the activity of calcineurin. That means thatthe effect of CuZnSOD is not due to the function of superoxide dismutasein redox regulation. These results teach that superoxide dismutaseinteracts physiologically with calcineurin and that CuZnSOD is onecomponent of the calcineurin pathway which is important for thephysiological functions of calcineurin.

These results are used to develop a new screening system for modulatorsof calcineurin in order to find inhibitors or activators of thecalcineurin signal pathway. The inventive method is based on the complexformation between calcineurin and superoxide dismutase in the presenceof potential modulators of this physiological interaction. If apotential modulator disturbs the complex formation, this substance is agood canditate for inhibiting the calcineurin signal pathway and couldpossibly be used as immunosuppressive drug, for example. On the otherhand it could be favourable to identify a substance which promotescomplex formation and thereby stimulates the calcineurin signal pathway,e.g. the T cell response in result. Such a substance could be used inorder to strengthen immune response. By the term “modulator” is meantany substance which influences the complex formation relating to theinventive method. Additionally is meant any substance which influencesthe interaction between calcineurin and its substrates, e.g. the peptideRII. Furthermore is meant any substance which influences the superoxidedismutase and/or calcineurin on the transcriptional, the translationaland/or the posttranslational level.

Calcineurin as used in the inventive method is build up by theregulatory subunit A and the catalytic subunit B. The presence of bothsubunits is essential for physiological activity of calcineurin.Nevertheless, it is possible to perform the inventive method using onlyone of the subunits. There are several isoforms of calcineurinconsisting of subunit calcineurin B and one out of the group comprisingsubunit calcineurin A-alpha, A-beta and A-gamma. Each isoform representsa special cell and tissue specific distribution. Therefore, the choiceof isoform could be crucial for cell and tissue specifity of thesubstance to be screened. With respect to clinical application of thesubstances to be screened preferably human forms of the proteins areused.

Furthermore it is preferred to perform the inventive method in thepresence of calmodulin and calcium, because the activity of calcineurinis dependent on these factors. Preferably the cytosolic form ofsuperoxide dismutase containing copper and zinc is used for complexformation, because interaction between the mitochondrial form ofsuperoxide dismutase containing e.g. manganese normally does not occurunder physiological conditions. The complex formation is performed inthe presence of at least one potential modulator of calcineurin or thecalcineurin signal pathway, respectively. The complex comprisingcalcineurin A, calcineurin B, superoxide dismutase and preferablycalmodulin is the target for potential modulators which could stabilizeor disturb the complex.

Advantageously, the complex formation is monitored during the wholeprocess. It is possible to add the modulator before or after the complexformation has been performed. Preferably the modulator is added beforecomplex formation because the effect of a weak modulator will possiblynot be monitorable when complex formation has already finished.

In principle, there are two possibilities to monitor the complexformation. Firstly, the complex formation is directly monitored by theuse of labeled components in the complex, preferably by fluorescencedetection. Secondly, the complex formation is monitored by the activityof the complex, especially the enzymatic activity of calcineurin. Thissecond method can be performed in addition to the firstly mentionedmethod or as an alternative. Clearly, the inventive method is notrestricted by the method for detecting the influence of the modulator oncomplex formation.

In a preferred embodiment of the invention, a mixture of substancescomprising at least one potential modulator is analyzed by the inventivemethod. By isolating the complex together with the possibly interactingmodulator it is possible to separate the modulator out of the mixtureand to identify it by common methods.

In one preferred embodiment of the invention the calcineurin and/orsuperoxide dismutase are labeled. Especially preferred is the use offluorescent labels. Preferably, the labeled proteins are fusion proteinscomprising a fluorescent protein, e.g. enhanced green fluorescentprotein (EGFP). These fusion proteins are provided by geneticengineering methods. It is also possible to label said proteins by othermethods known to experts in the art, e.g. by the use of radioactiveisotopes which are incorporated into the proteins.

Advantageously the components of the complex, i.e. calcineurin andsuperoxide dismutase are expressed in the cell, especially in aneukaryotic cell, as fluorescent fusion proteins. By the use of laserfluctuation correlation spectroscopy the complex formation of labeledproteins is monitored directly within the cell. This embodiment of theinvention is described in greater detail in the example. The inventioncomprises several vectors useful for the expression of calcineurinand/or superoxide dismutase in eukaryotic cells. These vectors encodethe proteins, especially CuZnSOD and the different subunits ofcalcineurin, as fusion proteins in connection with the fluorescentprotein EGFP (enhanced green fluorescent protein). EGFP is only oneexample of possible labels useful in respect of the inventive method.Furthermore, the invention comprises cells, especially eukaryotic cells,stably transfected with the above-mentioned vectors thereby expressingsuperoxide dismutase and/or calcineurin. Preferably, these proteins arecoexpressed, i.e. expressed within the same cell.

In an especially preferred embodiment, the genetic information of fusionproteins is integrated in the cell by homologous recombination. Thatmeans that the gene encoding the recombinant protein, especially thefluorescent fusion protein, is incorporated in the genome of the cell inthe place of the naturally occuring gene. This leads to a cellessentially lacking the natural protein. By the use of such cells it ispossible to identify modulators by the inventive method which influencethe transcriptional, translational or posttranslation level ofcalcineurin and/or superoxide dismutase expression.

In another embodiment of the inventive method the components of thecomplex are isolated and preferably purified before complex formation isperformed in vitro. Advantageously, the proteins are provided with a tagin order to facilitate purification, e.g. a histidine (his) tagconsisting of several histidines in sequence which permits affinitypurification by known procedures. Corresponding vectors encoding thetagged proteins are comprised by the invention. These vectors areespecially useful as prokaryotic expression vectors. Furthermore, theinvention comprises cells bearing said vectors.

Advantageously, following purification of the his-tagged proteins thetag is excised by appropriate enzymatic digestion, e.g. by the use ofcathepsin-C of carboxypeptidase-A. Especially preferred is thepurification of calcineurin by ferro-nitrilotricacetat-metal (Fe-NTA)affinity chromatography and the purification of superoxide dimutase bycopper/zinc-nitrilotriacetat-metal (CuZn-NTA) affinity chromatography.Nevertheless, other purification procedures known to experts in the artare possible. Natural occuring protein could also be used in theinventive manner.

Besides the use in purification of calcineurin and/or superoxidedismutase Ni (nickel)-NTA, Fe-NTA and/or Cu/Zn-NTA is used to immobilizethe his-tagged calcineurin and/or superoxide dismutase in order toisolate naturally occuring ligands of these proteins using thisinventive matrix. By the term “ligand” is meant any low- orhighmolecular endogenous, exogenous or synthetic substance whichinteracts with said proteins. This could be a peptide, protein,carbohydrate, lipid, nucleic acid or a synthetic polymer, for example.These so-identified ligands are potential candidates for modulators ofthe calcineurin signal pathway.

When performing complex formation in vitro it could be preferred to addcalmodulin and/or calcium to the reaction because these factors arenecessary for enzymatic activity of calcineurin.

In another preferred embodiment of the invention the complex formationis monitored indirectly by analyzing the enzymatic activity ofcalcineurin. As outlined above the phoshatase activity of calcineurin isstrictly dependent on the interaction with superoxide dismutase.Therefore, it is possible to monitor the complex formation indirectly bythe measurement of phosphatase activity according to standardprocedures. This is especially preferred if the laboratory equipment toperform fluorescent measurements as described above is not available.Furthermore, enzymatic analysis could be used in addition tofluorescence detection like laser fluctuation correlation spectroscopy,e.g. as control.

Preferably the enzymatic activity is analyzed by the use of a labeledsubstrate of calcineurin. The substrate is preferably labeled byfluorescence. One especially preferred substrate is the peptide RIIcharacterized by the sequence, SEQ ID 35:

-   -   Asp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Ala-Ala-Glu.

In a preferred embodiment this peptide carries a fluorescent label atserine in position 15. This amino acid is labeled with fluorescein byincubating the peptide with fluroescein-phosphoamidit, thereby providinga labeled substrate (RII-Fluophos). RII interacts with the active centerof calcineurin, but it is not converted by the phosphatase. Hereby it ispossible to label calcineurin in the active state. Furthermore, it ispossible to phosphorylate RII-Fluophos at the fluorescein moiety asdepicted below at tyrosine residues. Due to the phosphorylationRII-Fluophos loses its fluorescence and thereby provides a phosphatasesubstrate which becomes fluorescent subsequent to dephosphorylation.

This peptide could be provided synthetically or it is expressed bycells, especially eucaryotic cells, which have been transfected withappropriate vectors encoding said peptide or other peptides useful asphosphatase substrates. In one embodiment of the invention thefluorescent peptide is used as peptide label in fluorescence microscopy.This provides another method in order to analyze the active state of thecalcineurin/superoxide complex.

The inventive method as outlined above is suitable for developing ahigh-throughput bioassay to identify inhibitors and/or activators of thecalcineurin signal pathway. Details are described in the example.

The invention comprises the use of inhibitors of activators of thecalcineurin signal pathway for the treatment of acute and/or chronicneurological and cardiovascular diseases like Alzheimer, Parkinson,epilepsy, ischemia and heart-failure. Furthermore the use asimmunosuppressive drugs, e.g. in the field of transplantation surgeryand inflammatory diseases is included.

Finally the invention comprises a kit for screening of modulators ofcalcineurin. The kit provides calcineurin and superoxide dismutaseenabling complex formation for the screening for modulators ofcalcineurin as described above. In a first embodiment of the kit thecomponents of the complex are provided as proteins. This kit is suitablefor performing the inventive method in vitro. In a second embodiment ofthe kit the proteins are provided in the form of vectors. These vectorshave to be transformed/transfected into cells leading to the expressedproteins. These vectors are prokaryotic or eukaryotic expressionvectors, respectively, and could be used to produce the proteins for thein vitro assay or for the assay using complete cells as described above.In a third embodiment of the inventive kit cells transformed/transfectedwith the said vectors are provided saving the step oftransforming/transfecting for the user. For details of the inventive kitreference is made to the above description.

The new approach to identify new substance classes ofcalcineurin/CuZnSOD inhibitors comprises inter alia:

-   -   coexpression of CuZnSOD/calcineurin A and calcineurin B to        generate a oxidative stable enzyme which is suitable for drug        screening,    -   efficient purification of CuZnSOD on CuZn-nitrilotriacetat-metal        affinity chromatography to retain enzymatic activity,    -   efficient purification of calcineurin on        Fe-nitrilotriacetat-metal affinity chromatography to retain        enzymatic activity and prohibit Fe²⁺ oxidation,    -   identifying that mutations in the CuZnSOD associated with a        neurological disorder (amyotrophic lateral sclerosis) are also        critical for calcineurin-CuZnSOD interaction,    -   use of fluorescent labeled recombinant CuZnSOD and calcineurin        to screen for CuZnSOD/calcineurin activators or inhibitors,    -   use of fluorescent labeled RII-peptide and calcineurin to screen        for calcineurin activators or inhibitors,    -   identification of calcineurin/CuZnSOD inhibitors or activators        by using the recombinant enzymes as affinity ligands to purify        new drugs from natural sources,    -   inclusion of all isoforms, all known and two newly identified        splicevariants into the screening procedure, which allows the        identification of less toxic and tissue specific drugs which are        more suitable for the therapeutical treatment of different        clinical indications.

The described features of the invention and further features result ingreater detail from the examples in combination with the subclaims. Thefeatures could be realized in combination with each other or alone.

EXAMPLE

1. Cloning of CuZnSOD transcript from human brain poly-A-RNA—Cloning ofhuman CuZnSOD was performed by reverse transcription PCR using humanbrain poly-A-RNA as template (Clontech, Palo Alto, Calif., USA). Theoligonucleotides SODs1 5′-ttc cgt tgc agt cct cgg aac-3′, SODas1 5′-taaggg gcc tca gac tac atc-3′, SOD-PQE60s2 5′-caa gcc atg gcg acg aag gccgtg tgc gtg ctg-3′, SOD-PQE60 as2 5′-gaa gat ctt tgg gcg atc cca att acacca c-3′, SOD-PQE30-s2 5′-cgc gga tcc gcg acg aag gcc gtg tgc gtg-3′ andSOD-PQE30-as2 5′-ggg ttc gaa tta ttg ggc gat ccc aat tac-3′ weresupplied by Interactiva (Ulm, Germany). Reverse transcription wasperformed with the SODas1 primer and 100 ng of poly-A-RNA according tothe manufacturer's protocol (Expand reverse transcriptase, BoehringerMannheim, Germany). The human CuZnSOD cDNA was amplified by nested PCR.The first PCR was performed in 20 μl, using 0.5 μl reverse transcriptionproduct, 10 μM SODs1 and SODas1 primers, 300 μM dNTPs, 2 μl of themanufacturer's 10×PCR buffer and 2.5 U Taq-polymerase with 30 cycles of1 min 95° C., 1 min 45° C., 1 min 72° C. followed by a second PCR (50μl) with 5 μl of the purified first PCR product, 10 μM SOD-PQE60s2 andSOD-PQE60 as2 primers, 300 μM dNTPs, 5 μl of the manufacturer's PCRbuffer and 2.5 U Taq-polymerase with 30 cycles of 1 min 95° C., 1 min60° C., 1 min 72° C. (Taq-polymerase, Pharmacia Biotech, Uppsala,Sweden). For the subcloning into pQE30 expression vector the primersSOD-PQE30-s2 and SOD-PQE30-as2 were used instead ofSOD-PQE60s2/SOD-PQE60as2.

2. Subcloning of human CuZnSOD into pQE60 expression vector (C-terminalfusion protein)—The SOD-pQE60 PCR product was purified by gel extractionprior to NcoI/BglII restriction (New England Biolabs). In order togenerate a C-terminal histidine tag fusion protein the CuZnSODtranscript was ligated into the NcoI/BglII treated prokaryoticexpression vector pQE60 (QIAexpress expression kit type IV and type ATG,Qiagen, Hilden, Germany). For selection, amplification and sequencing ofthe CuZnSOD vector construct (CuZnSOD-pQE60), A1 ligation product wastransformed into E. coli M15[pREP4] cells (QIAexpress expression kittype ATG, Qiagen, Hilden, Germany). Correct reading frames and exclusionof mismatches were confirmed by radioactive and automated sequencing onboth strands (T7-sequencing kit, Pharmacia Biotech, Uppsala, Sweden; ABI377 sequencer, Applied Biosystems, USA).

3. Subcloning of human CuZnSOD into pQE30 expression vector (N-terminalfusion protein)—The SOD-pQE30 PCR product was purified by gel extractionprior to direct ligation into the pCR2.1 vector according to themanufacturer's protocols (TA-Cloning Kit, Invitrogen, De Schelp,Netherlands). After amplification and plasmid purification thepCR2.1-CuZnSOD vector construct was restricted with BamHI to yield aCuZnSOD transcript extended at the 3′-end with the sequence5′-GAATTCCAGCACACTGGCGGCCGTTACTAGTGGATCC-3′ which originates from PCR2.1vector and includes additional EcoRI/BstX-I/SpeI/BamHI restrictionsites. In order to generate a N-terminal histidine tag fusion proteinthe extended transcript was ligated into the BamHI/HindIII treatedprokaryotic expression vector pQE30 (QIAexpress expression kit type IV,Qiagen, Hilden, Germany), blunted by incubation withKlenow-DNA-polymerase and circularized by a second treatment withT4-DNA-Ligase (Boehringer Mannheim, Germany). For selection,amplification and sequencing of the CuZnSOD vector construct(CuZn-SOD-pQE30), 10 μl ligation product was transformed into E. coliM15[pREP4] cells (QIAexpress expression kit type IV and type ATG,Qiagen, Hilden, Germany). Correct reading frames and exclusion ofmismatches were confirmed by radioactive and automated sequencing onboth strands (T7-sequencing kit, Pharmacia Biotech, Uppsala, Sweden; ABI377 sequencer, Applied Biosystems, USA).

4. Site directed mutagenesis (point mutations associated with theneurological disorder Amyotrophic Lateral Sclerosis and important forcalcineurin/CuZnSOD protein interaction)—Amino acid substitutions wereintroduced according to the manufacturer's protocol, using the primersSOD-PQE60-A4V (5′-caa gcc atg gcg acg aag gtc gtg-3′), SOD-A4V (5′-tcbgcg acg aag qtc gtg tgc gtg ctg-3′), SOD-G37R (5′-gg aag catt aaa agactg act gaa ggc-3′), SOD-D90A (5′-aat gtg act gct gcc aaa gat ggtgtg-3′), SOD-G93A (5′-gct gac aaa gat gct gtg gcc gat gtg-3′),SOD-AflIII (5′-acg cag gaa aga aca tgt gag caa aag-3′), SOD-BglII(5′-acg cag gaa aga aga tct gag caa aag-3′) and the expression vectorCuZnSOD constructs CuZn-SOD-pQE30 and CuZnSOD-pQE60, respectively(Chameleon site directed mutagenesis kit, Stratagene, San Diego, Calif.,USA). Incorporation of the site-directed mutations was confirmed by DNAsequencing of the expression vector. Site directed mutagenesis yieldedeight additional vector sequences corresponding to eight proteinsequences with clinical relevant amino acid substitutions:

amino acid subst. nucleic acid subst. Vector-construct (pos. in protein)(pos. in sequ. prot.) CuZnSOD-pQE60 WT = wild-type = SEQ ID NO 15CuZnSOD-pQE60-(A4V) Ala-4 -> Val-4 c-128 -> t-128 CuZnSOD-pQE60-(G37R)Gly-37 -> Arg-37 g-226 -> a-226 CuZnSOD-pQE60-(D90A) Asp-90 -> Ala-90a-386 -> c-386 CuZnSOD-pQE60-(G93A) Gly-93 -> Ala-93 g-395 -> c-395CuZnSOD-pQE30 WT = wild-type = SEQ ID NO 13 CuZnSOD-pQE30-(A4V) Ala-4 ->Val-4 c-161 -> t-161 CUZnSOD-pQE30-(G37R) Gly-37 -> Arg-37 g-259 ->a-259 CUZnSOD-pQE30-(D90A) Asp-90 -> Ala-90 a-419 -> c-419CuZnSOD-pQE30-(G93A) Gly-93 -> Ala-93 g-428 -> c-428

5. Recombinant expression and purification of wild-type and mutatedCuZnSOD—The CuZnSOD-pQE60 or CuZnSOD-pQE30 vector transformed E. coliM15[pREP4] cells were plated on LB/ampicillin (100 μg/ml)/kanamycin (25μg/ml) agar. Expression cultures were grown in 250 ml LB/ampicillin (100μg/ml)/kanamycin (25 μg/ml) until the OD₆₀₀ was 0.6. Constitutiveleakage expression of human CuZnSOD was fully prevented by the repressorplasmid pREP4-lacI. Production of the human CuZnSOD fusion proteins wasinduced by addition of IPTG (1 mM). After two hours the bacterial cellswere harvested by centrifugation (4000 g, 20 min), resuspended in 8 mlbuffer A (20 mM Tris-HCl pH 7.9, 5 mM imidazole, 500 mM NaCl) andhomogenized by three freeze thaw cycles and sonication on ice (Bandelinsonoplus GM70, 300 W, 3×10 sec). The lysate was centrifuged (10.000 g,20 min) and incubated with 750 μl CuZn-NTA (nitrilotriacetat)-agarosefor batch affinity binding for 1 h at 4° C. (Qiagen expressionist kit,Qiagen, Hilden, Germany). CuZn-NTA-agarose was prepared fromNi-NTA-agarose (Qiagen expressionist kit, Qiagen, Hilden, Germany) bysubsequent washes in:

-   1) 2 volumes of bidistilled water-   2) 3 volumes of regeneration buffer (6 M guanidiniumhydrochloride,    0.2 M acetic acid)-   3) 5 volumes bidistilled water-   4) 3 volumes 2% SDS-   5) 1 volume 25% ethanol-   6) 1 volume 50% ethanol-   7) 1 volume 75% ethanol-   8) 5 volumes 100% ethanol-   9) 1 volume 75% ethanol-   10) 1 volume 50% ethanol-   11) 1 volume 25% ethanol-   12) 1 volume bidistilled water-   13) 5 volumes 100 mM Na-EDTA pH 8.0-   14) 5 volumes bidistilled water-   15) 2 volumes 100 mM CUS04/100 mM ZnS04 1 mM reduced glutathione/1    mM dithiothreitol-   16) 2 volumes bidistilled water-   17) 2 volumes regeneration buffer (6 M guanidiniumhydrochloride, 0.2    M acetic acid)-   18) 2 volumes buffer buffer A2 (20 mM Tris-HCl pH 7.9, 5 mM    imidazole, 500 mM NaCl, 200 μM CuS04/200 μM ZnS04/1 mM reduced    glutathione/1 mM dithiothreitol.

The batch was applied to a 30 ml chromatography column, washed with 15ml buffer A (20 mM Tris-HCl pH 7.9, 5 mM imidazole, 500 m.M NaCl) andsubsequently with 8 ml buffer B (20 mM Tris-HCl pH 7.9, 60 mM imidazole,500 mM NaCl). C-terminal or N-terminal histidine tagged CuZnSOD waseluted three times with 1.2 ml buffer C (10 mM Tris-HCl, 500 mMimidazole, 250 mM NaCl). Purity and correct expression products werechecked by immunoblotting or N-terminal protein sequencing afterseparation of 20 μl eluate in SDS-PAGE (discontinuous 12.5% SDS-PAGE).To examine the protein levels in bacterial culture all CuZnSOD variantswere induced synchronously at OD₆₀₀=0.6 With 1 mM IPTG. After 1 h, 2 h,3 h, 4 h and 20 h, aliquots (1 ml) of E. coli cultures were taken,centrifuged and homogenized in buffer A as described. The pellet wasresuspended in 1 ml H₂O. Subsequently, 20 pl of the supernatant (solublefractions) or 20 μl of the sonicated pellet suspension (insolublefractions) were mixed with 7 μl of denaturing sample buffer (10% SDS,10% mercaptoethanol, 20% glycerol, 130 mM Tris-HCl pH 6.8, 0.03%bromphenol blue). The samples were heated for 2 minutes at 80° C. andanalyzed by 12% SDS-PAGE. After coomassie staining, theelectropherograms were digitized with a CCD camera (Gel Doc 1000,BioRAD) and analyzed by densitometry using NIH-Image software (1.61).

6. Processing of CuZnSOD—In order to remove the nonphysiologicalhistidine tag and to yield CuZnSOD useful for clinical applications theN-terminal histidin tagged CuZnSOD was proteolytically processed withcathepsin-C or the C-terminal variant was processed withcarboxypeptidase-A according to the manufacturer's protocols(Boehringer-Mannheim, Mannheim, Germany). Treatment with cathepsin-Cyielded a processed CuZnSOD starting with the amino acids NH₂-GSATKAVCVLKGDGP (indicated in sequence protocol CuZnSOD-pQE30 SEQ ID NO 13).C-terminal fusion protein was yielded the C-terminal amino acid sequenceVIGIAQR-COOH (indicated in sequence protocol CuZnSOD-pQE30 SEQ ID NO13). Verification was done by peptide sequencing.

7. Reactivation of CuZnSOD—In order to yield physiologically relevantactive homodimeric CuZnSOD, the CuZn-NTA eluate was ultrafilteredthrough a 5 kD membrane (omegacell, Filtron, Northborough, Mass., USA).For buffer exchange the samples were washed three times inreconstitution buffer (50 mM sodium citrate pH 5.5, 1 mM DTT). Theprotein solutions were incubated at 8° C. for 7 days (250 μg/mlprotein). After distinct time intervals aliquots of the refoldingmixture were either analyzed by native gel electrophoresis (2.6 μgCuZnSOD) and activity staining or assayed in a spectrophotometer (0.5-1Ag CuZnSOD, superoxide dismutase assay kit, Calbiochem, San Diego,Calif., USA). For visualization of protein bands native gels werestained with coomassie blue. For the production of larger CuZnSODamounts M15-E. coli cells were subsequently grown in 15 ml, 200 ml, 2500ml and 20 L flasks. Refolded CuZnSOD proteins were dialyzed against 100volumes of buffer D (10 mM Tris-HCl 0.1% Saccharose) and lyophylized.

8. SOD assay and activity staining—Enzymatic activity of the CuZnSODproteins were either analyzed by 10% native gel electrophoresis andactivity staining with nitrotetrazolium blue dye or by a quantitativespectrophotometrically assay according to published protocols (Beauchampand Fridovich, 1971; Nebot et al., 1993). Protein yields were determinedby the Bradford method (Protein assay kit, BioRAD, Hercules, Calif.,USA). The concentration of purified CuZnSOD was determinedspectrophotometrically using the extinction coefficient 265=1.84×10⁴ M⁻¹cm⁻¹.

9. Subcloning of human CuZnSOD into pEGFP eukaryotic expression vectorand generation of stable transfected PC12 cells (C-terminal fusionprotein with enhanced green fluorescent protein as a fluorescentmarker/label)—Using 10 μM of the primers SOD-pEGFP-s 5′-ccg cgg gcc cgccat ggc gac gaa ggc cgt gtg cgt gc-3′ and SOD-pEGFP-as 5′-gct cac catggt ggt ttg ggc gat ccc aat tac acc α-3′, 10 ng CuZnSOD-pQE60 vector,300 μM dNTPs, 5 μl of the manufacturer's PCR buffer and 2.5 UTaq-polymerase with 25 cycles of 1 min 95° C., 1 min 60° C., 1 min 72°C. (50 μl total volume, Taq-polymerase, Pharmacia Biotech, Uppsala,Sweden) a PCR product was generated which was cleaved by ApaI/NcoIdigestion. The purified PCR product was ligated into ApaI/NcoI treatedpEGFP—N3 vector (Clontech Laboratories, Palo Alto, Calif., USA). Afteramplification in XL2-Blue cells (25 μg/ml kanamycin) and plasmidpurification the CuZnSOD-pEGFP vector construct was transfected intoPC12 rat adrenal pheochromocytoma cells using the CalPhos™ TransfectionKit according to the manufacturer's protocols (Clontech Laboratories,Palo Alto, Calif., USA). Stable transfected CuZnSOD-pEGFP clones wereselected by fluorescence microscopy (exitation 488 nm/emission 520 nm,MRC 1024 confocal microscope, BioRAD Laboratories, Hercules, Calif.,USA).

10. Cloning of the regulatory subunit human calcineurin-B—Cloning ofhuman calcineurin-B was performed by reverse transcription PCR usinghuman brain poly-A-RNA as template (Clontech, Palo Alto, Calif., USA).The oligonucleotides CNBa-s1 5′-ccg ccg acc cgc cga gca-3′, CNBa-as15′-ggt act ctc tga taa gag-3′, CNBa-s3 5′-gga att ccc cgg gga aag aggaga aat taa cta tgg gaa atg agg caa gtt atc-3′, CNBa-as2 5′-ttc cgg gcccaa gct tct aat taa tca cac atc tac cac cat c-3′ were supplied byInteractiva (Ulm, Germany). Reverse transcription was performed with theCNBa-as1 primer and 100 ng of poly-A-RNA according to the manufacturer'sprotocol (Expand reverse transcriptase, Boehringer Mannheim, Germany).The human calcineurin-B cDNA was amplified by nested PCR. The first PCRwas performed in 20 μl, using 0.5 μl reverse transcription product, 10μM CNBa-s and CNBa-as1 primers, 300 μM dNTPs, 2 μl of the manufacturer's10×PCR buffer and 2.5 U Pfu-polymerase with 20 cycles of 1 min 95° C., 1min 55° C., 2 min 72° C. followed by a second PCR (50 μl) with 5 μl ofthe purified first PCR product, 10 μM CNBa-s3 and CNBa-as2 primers, 300μM dNTPs, 5 μl of the manufacturer's PCR buffer and 2.5 U Pfu-polymerasewith 20 cycles of 1 min 95° C., 1 min 55° C., 1 min 72° C.(Pfu-polymerase, Stratagene, San Diego, Calif., USA).

11. Cloning of the catalytic subunit human calcineurin-A-Alpha andsplice variants—Cloning of human calcineurin-A-alpha was performed byreverse transcription PCR using human brain poly-A-RNA as template(Clontech, Palo Alto, Calif., USA). The oligonucleotides CNAa-s1 5′-gcgtcg ctg tcc tcc ggc agc-3′, CNAa-as1 5′-gtg aac agg aag tgg tca ctg-3′,CNAa-s2 5′-cat gcc atg gatc cat gtc cga gcc caa ggc-3′, CNAa-as4 5′-tccccc cgg ggta ccc tag tta atc act gaa tat tgc tgc tat tac-3′ weresupplied by Interactiva (Ulm, Germany). Reverse transcription wasperformed with the CNAa-as1 primer and 100 ng of poly-A-RNA according tothe manufacturer's protocol (Expand reverse transcriptase, BoehringerMannheim, Germany) The human calcineurin-A-Alpha cDNA was amplified bynested PCR. The first PCR was performed in 25 μl, using 0.5 μl reversetranscription product, 10 μM CNAa-s1 and CNAa-as1 primers, 200 μM dNTPs,2.5 μl of the manufacturer's 10×PCR buffer and 1.25 U Pfu-polymerasewith 30 cycles of 40 seconds at 95° C., 40 seconds at 55° C., 3 min 72°C. followed by a second PCR (25 μl) With 2.5 μl of the purified firstPCR product, 10 μM CNAa-s2 and CNAa-as2 primers, 200 μM dNTPs, 2.5 μl ofthe manufacturer's PCR buffer and 2.5 U Pfu-polymerase with 25 cycles of40 seconds at 95° C., 40 seconds at 55° C., 3 min 72° C.(Pfu-polymerase, Stratagene, San Diego, Calif., USA).

Hereby a new splice variant was identified, which is important forcalcium regulation and proteolytic regulation of calcineurin-A. Thesplice variant lacks the hole catalytic phosphatase domain and part ofcalcineurin-binding-site (Elimination of nucleic bases 208-1317 insequence protocol CNAa1-pQE30 SEQ ID NO 17). The corresponding vector isnamed CNAa3-pQE30:

Location/Qualifiers

-   151 . . . 606/note=“splicevariant: Calcineurin A alpha 1 lacking    phosphatase domain, newly generated N-terminus exhibits protease    activity”-   115 . . . 150/note=“His-Tag”-   649 . . . 1161/note=“Calcineurin B;Calcineurin B alpha Ca²⁺ binding”

12. Cloning of the catalytic subunit human calcineurin-A-Beta and splicevariants—PCR was performed as described under 11. with the exceptionthat the primers CNAb-s1 5′-gag cct agc cga gcc ccg gg-3′ and CNAb-as15′-ctg gga agt agt ggg tca ctg-3′ were used for the first PCR and theprimers and CNAb-s2 5′-cat gcc atg gat cca tgg ccg ccc cgg agc c-3′ andCNAb-as4 5′-tcc ccc cgg ggt acc cta gtt aat cac tgg gca gta tgg ttg ccag-3′ were used for second PCR.

13. Cloning of the catalytic subunit human calcineurin-A-Gamma andsplice variants—PCR was performed as described under 11. with theexception that the primers CNAg-s1 5′-gga gcC tgg agg agg ccg ag-3′ andCNAg-as1 5′-cgg cag gac tct aag tca tga-3′ were used for the first PCRand the primers and CNAg-s2 5′-cat gcc atg gat cca tgt ccg gga ggc gcttc-3′ and CNAg-as4 5′-tcc ccc cgg ggt acc cta gtt aat cat gaa tqg gctttc ttc cct t-3′ were used for second PCR.

Hereby a new splice variant was identified, which is important forcalcium regulation and proteolytic regulation of calcineurin-A. Thesplice variant with human exon is not yet available in gene database(substitution of nucleic bases 1474-1503 in sequence protocolCNAg2-pQE30 SEQ ID NO 32) with 5′-ACA GTA GAA GCG GTA GAG GCC CGG GAAGCC-3′ (corresponding peptide: NH2-TVEAVEAREA-COOH). The correspondingvector is named CNAg3-pQE30.

Location/Qualifiers 115 . . . 150 /note = “His-Tag” 151 . . . 1689 /note= “Calcineurin-A-Gamma-2” 1474 . . . 1503 /note = “human braincalcineurin-A-gamma alter- native exon = interaction domain withcytoskelett, death-domain homolog, stomatin homolog” 1690 . . . 1731/note = “RBS&MCS2” 1732 . . . 2244 /note = “Calcineurin-B”

14. Subcloning of calcineurin-B and calcineurin-A variants intopQE30—For the recombinant expression in procaryotic cells calcineurin-Bwas subcloned with either calcineurin-A-alpha1, calcineurin-A-alpha2,calcineurin-A-beta1, calcineurin-A-beta2, calcineurin-A-gamma1 orcalcineurin-A-gamma2. The purified calcineurin-A-alpha,calcineurin-B-alpha or calcineurin-A-gamma PCR products (described in11.-13.) were restricted with BamHI/XmaI. The purified calcineurin-Bproduct (described in 10.) was restricted with XmaI/HindIII and ligatedtogether with the respective calcineurin-A-fragment into theBamHI/HindIII treated vector pQE30 to yield the final procaryoticexpression vector constructs CNAa1-pQE30, CNAa2-pQE30, CNAa3-pQE30,CNAb1-pQE30, CNAb2-pQE30, CNAg1-pQE30, CNAg2-pQE30 and CNAg3-pQE30.

15. Recombinant coexpression and purification ofcalcineurin-B/calcineurin-A heterodimers with CuZnSOD—CNAa1-pQE30,CNAa2-pQE30, CNAa3-pQE30, CNAb1-pQE30, CNAb2-pQE30, CNAg1-pQE30,CNAg2-pQE30 or CNAg3-pQE30 were transformed into E. coliM15[pREP4][CuZnSOD-pQE30] to yield cells able to coexpresscalcineurin-A, calcineurin-B and CuZnSOD. cells were plated onLB/ampicillin (100 μg/ml)/kanamycin (25 μg/ml) agar. Expression cultureswere grown in 250 ml LB/ampicillin (100 μg/ml)/kanamycin (25 μg/ml)until the OD₆₀₀ was 0.6. Constitutive leakage expression was preventedby the repressor plasmid pREP4-lacI. Production of the humancalcineurin-A/calcineurin-B histidine tagged heterodimers was induced byaddition of IPTG (1 mM). After four hours the bacterial cells wereharvested by centrifugation (4000 g, 20 min), resuspended in 8 ml bufferA (20 mM Tris-HCl pH 7.9, 5 mM imidazole, 500 mM NaCl) and homogenizedby three freeze thaw cycles and sonication on ice (Bandelin sonoplusGM70, 300 W, 3×10 sec). The lysate was centrifuged (10,000 g, 20 min)and incubated with 750 μl Fe-NTA-agarose for batch affinity binding for1 h at 4° C. (Qiagen expressionist kit, Qiagen, Hilden, Germany).Fe-NTA-agarose was prepared from Ni-NTA-agarose (Qiagen expressionistkit, Qiagen, Hilden, Germany) by subsequent washes in:

-   1) 2 volumes of bidistilled water-   2) 3 volumes of regeneration buffer (6 M guanidiniumhydrochloride,    0.2 M acetic acid)-   3) 5 volumes bidistilled water-   4) 3 volumes 2% SDS-   5) 1 volume 25% ethanol-   6) 1 volume 50% ethanol-   7) 1 volume 75% ethanol-   8) 5 volumes 100% ethanol-   9) 1 volume 75% ethanol-   10) 1 volume 50% ethanol-   11) 1 volume 25% ethanol-   12) 1 volume bidistilled water-   13) 5 volumes 100 mM Na-EDTA pH 8.0-   14) 5 volumes bidistilled water-   15) 2 volumes 100 mM FeSO₄/1 mM reduced glutathione 1 mM    dithiothreitol/100 mM ascorbic acid-   16) 2 volumes bidistilled water-   17) 2 volumes regeneration buffer (6 M guanidiniumhydrochloride, 0.2    M acetic acid)-   18) 2 volumes buffer buffer A3 (20 mM Tris-HCl pH 7.9, 5 mM    imidazole, 500 mM NaCl, 200 gM FeSO₄/1 mM reduced glutathione/1 mM    dithiothreitol/1 mM ascorbic acid.

The batch was applied to a 30 ml chromatography column, washed with 15ml buffer A4 (20 m.M Tris-HCl pH 7.9, 5 mM imidazole, 500 mM NaCl/1 mMreduced glutathione/1 mM dithiothreitol/1 mM ascorbic acid) andsubsequently with 8 ml buffer B (20 m.M Tris-HCl pH 7.9, 60 mMimidazole, 500 mM NaCl/1 mM reduced glutathion/1 mM dithiothreitol/1 mMascorbic acid). N-terminal histidine tagged calcineurin-A/calcineurin-Bheterodimer was eluted three times with 1.2 ml buffer C (10 mM Tris-HCl,500 mM imidazole, 250 mM NaCl/1 mM reduced glutathion/1 mMdithiothreitol/1 mM ascorbic acid, buffer was degased and subsequentlysaturated with nitrogen). To prevent oxidation of calcineurin, theeluate was stored at −80° C. in nitrogen containing and oxygen freevials. Purity and correct expression products were checked byimmunoblotting or N-terminal protein sequencing after separation of 20μl eluate in SDS-PAGE (discontinuous 12, 5 SDS-PAGE).

16. Subcloning of human calcineurin-A-Alpha into pEGFP eukaryoticexpression vector and generation of stable transfected PC12 cells(C-terminal fusion protein with enhanced green fluorescent protein as afluorescent marker)—The vector CNAa2-pQE30 was digested with BamHI/XmaIto generate a sticky end CNAa2 fragment. The purified fragment wasligated into Bgl-II/XmaI treated pEGFP-Cl vector (Clontech Laboratories,Palo Alto, Calif., USA). After amplification in XL2-Blue cells (25 μg/mlkanamycin) and plasmid purification the CNAa-pEGFP vector construct wastransfected into PC12 rat adrenal pheochromocytoma cells using theCalPhos™ Transfection Kit according to the manufacturer's protocols(Clontech Laboratories, Palo Alto, Calif., USA). Stable transfectedCNAa-pEGFP clones were selected by fluorescence microscopy during athree month propagation procedure (exitation 488 nm/emission 520 nm, MRC1024 confocal microscope, BioRAD Laboratories, Hercules, Calif., USA).

17. Subcloning of calcineurin-A-Beta into pEGFP—The same procedure asdescribed in 16. was applied except that the CNAa2-pQE30 vector wassubstituted by CNAb2-pQE30 to generate CNAb-pEGFP.

18. Subcloning of calcineurin-A-Gamma into pEGFP—The same procedure asdescribed in 16. was applied except that the CNAa2-pQE30 vector wassubstituted by CNAg2-pQE30 to generate CNAg-pEGFP.

19. Western blotting and protein sequencing—Transfer of purifiedproteins from 12% SDS-PAGE to PVDF membranes (Boehringer-Mannheim,Mannheim, Germany) was performed according to standard protocols usingtransfer buffer (48 mM Tris, 39 mM Glycine, 20% methanol, 1% SDS, pH9.2) and following blotting conditions: 75 min at 25 V/110 mA. Blocking,washing and detection (HRP detection system) were performed according tothe manufacturer's protocols (ECL kit, Amersham, Buckinghamshire, UK).An anti-human CuZnSOD antibody (1:5,000 dilution, rabbit polyclonalanti-human SOD1 antibody; BIOMOL, Hamburg, Germany) was used as primaryantibody and an anti-rabbit IgG antibody (1:10,000 dilution) labeledwith HRP was used as secondary antibody. For the detection ofcalcineurin-A (alpha, beta, gamma isoforms) a polyclonal calcineurin-Aantibody was used as 1:5000 dilution (Sigma Aldrich, Deisenhofen,Germany). For N-terminal protein sequencing the PVDF membrane was soakedin 100% methanol. Proteins which seemed to be blocked by N-terminalposttranslational modifications were treated withacylamino-acid-peptidase according to the manufacturer's protocol(Boehringer-Mannheim, Mannheim, Germany). Coomassie brilliant bluestained bands were cut out. Automated Edman degradation of peptides wasperformed on an Applied Biosystems protein sequencer (476A).

20. Calcineurin phosphatase assay—100 ng-4 μg recombinant calcineurin(calcineurin-A/B heterodimer), 100 ng-1 μg purified bovine braincalcineurin (Sigma Aldrich, Deisenhofen, Germany) or 100 μg homogenizedtissue or cell extracts were used for classical calcineurin phosphataseassays. 100 μg cells or tissue were homogenized exactly as described(Stemmer et al., 1995). Partly purified and redox sensitive calcineurinwas prepared by centrifugation at 14,000 rpm at 4° C. for 10 min(Eppendorf centrifuge 5417R) and the resulting supernatant was separatedon a 1.5×10 cm Sephadex-G50 gelfiltration column as described (Stemmeret al., 1995), (Gold et al., 1997). Phosphotyrosine phosphatase assaywas performed in microplates (100 μl total assay volume) either using 30μM fluoresceinmonophosphate or 20 mM para-nitrophenylphosphate (SigmaAldrich, Deisenhofen, Germany); 10 μl recombinant, purified or partlypurified and assay buffer (25 mM Tris/HCl, pH 7; 2 mM CaCl₂; 0.1 μMcalmodulin; 25 μM FK506). After starting the enzymatic reaction withpara-nitrophenylphosphate or fluoresceinmonophosphate the absorbance at405 nm (para-nitrophenylphosphate) or fluorescence λ exication=485 nm; λemission=520 nm) was monitored over 20 min at 30° C. using aUV/VIS/fluorescence microplate photometer (Biolumin 960 kineticfluorescence/absorbance photometer, Molecular Dynamics). Phosphoserinephosphatase assay was performed as described (Hubbard and Klee, 1991),(Wang et al., 1996). In short: 40 μl recombinant or partly purifiedcalcineurin was mixed with test buffer (40 mM Tris/Hcl pH 8; 0.1 M KCl;0.4 mg/ml BSA; 0.67 mM DTT; 0.67 μM calmodulin; 1 μM FKBP bindingprotein; 0.5 μM ocadaic acid for inhibition of phophatase A1 and A2) andenzymatic reaction and calcium induced redox-inactivation of calcineurinstarted by addition of 20 μl substrate buffer (7.7 μM radioactivephosphorylated RII-peptid, 2.0 mM CaCl₂). The assay was performed induplicates and the addition of 1 μM FK506 or cyclosporine was used toverify calcineurin activity for each reading point. The protectiveeffect of CuZnSOD against redox inactivation of calcineurin wasdetermined by addition of 3 μg recombinant human wild-type or mutatedCuZnSOD (constant CuZnSOD protein) or addition of 1.67 units ofrecombinant human wild-type or mutated CuZnSOD (constant CuZnSODactivity). The reaction mixture was incubated for 2 min at 30° C. andstopped with 100 mM potassium phosphate/5% TCA. The reaction mixture waspassed through a 0.5 ml ion-exchange column (Dowex; AG 50W-X8, BioRad)and the unbound phosphate eluted with 0.5 ml water. The quantity ofreleased phosphate was determined by a scintillation counting.

An enzymatic protein phosphatase assay was established using thenonphysiological substrate fluoresceinmonophosphate (FMP). Assuming aMichaelis-Menten kinetic for FMP and using the Lineweaver-Burk methodfor analysis of kinetic data a K_(M) of 40 μM and a V_(max) of 400μmol/min was determined. The assay was applicable to calcineurin andmagnesium dependent proteinphosphatase 2C (data not shown, (Grothe etal., 1998)). The enzymatic activity is linear in the range of 12.5 pM to75 pM calcineurin. FMP is more sensitive than para-nitrophenylphosphate(pNPP). Neither FMP nor pNPP are useful to measure calcineurin activityin crude preparations by inhibition with the immunsuppressive drugsFK506 or cyclosporine (cell homogenate, partly purified calcineurin).Both substrates also failed to measure calcium inducedredox-inactivation of calcineurin or CuZnSOD mediated protection of thisinactivation. The inhibition assay also failed when calcium wassubstituted against other divalent cations (Ni²⁺, Mg²⁺). Only thephysiological relevant substrate could be used in an immunsuppressivedrug inhibitory assay (RII-peptide phosphopeptide). In the classicradioactive assay 95% inhibition with 1 μM FK506 or cyclosporine wasdetermined. It is concluded that inhibition of calcineurin activity byimmunosuppresive drugs needs larger molecular weight substrates thanpNPP and FMP. Furthermore it is concluded that redoxsensitivity islinked to phosphoserine phosphatase activity and therefore notdetectable with phosphotyrosine analoges like pNPP or FMP. Therecombinant human wild-type CuZnSOD and purified human erythrocyteCuZnSOD (Sigma Aldrich, Deisenhofen, Germany) were effective to protect50-100% of calcineurin after calcium induced redox inactivation. MutatedCuZnSOD proteins, associated with the severe neurological disorderamyotrophic lateral sclerosis, were less effective to protectcalcineurin against redox inactivation.

Protective Effect of CuZnSOD of Calcium Induced Inactivation ofCalcineurin

Percentage of FK506 inhibitable RII-phosphopeptide activity after 20 mincompared with the activity at 0 min

constant protein constant activity human CuZnSOD (3 μg) (1.67 U)erythrocyte wild-type 57 +/− 10% 57 +/− 10% (8330 U/mg) recombinantwild-type 70 +/− 33% 56 +/− 22% (6380 U/mg) recomb. mutation D90A 42 +/−17% 32 +/− 15% (4590 U/mg) recomb. mutation G93A 16 +/− 16% 21 +/− 22%(2130 U/mg) recomb. mutation A4V 22 +/− 27% 8 +/− 3% (1820 U/mg) control(no CuZnSOD) 9 +/− 7% 9 +/− 7% (0 U/mg)

The protective effect does not depend on CuZnSOD activity since higherprotein amounts of mutated CuZnSOD corresponding to a higher enzymaticactivity were even less effective in protection of calcineurin.

Therefore it is concluded that amino acid substitutions, associated withfamilial amyotrophic lateral sclerosis, are important for the proteininteraction of calcineurin and CuZnSOD and therefore are involved in theCuZnSOD mediated protection of calcium induced redox inactivation ofcalcineurin. Since this protective effect is disturbed in amyotrophiclateral sclerosis and protection of calcineurin by CuZnSOD it may alsobe important in other neurological and cardiovascular diseases(Alzheimer, Parkinson, epilepsy, ischemia, heart-failure).

An high-throughput bioassay was developed to detect and isolateartificial or endogenous drugs enhancing (activators)CuZnSOD-calcineurin interaction and therefore protecting calcineurinagainst redox-inactivation or drugs reducing (inhibitors)CuZnSOD-calcineurin interaction and therefore inhibit calcineurinactivity. Inhibitors are useful to substitute toxic immunsuppressivedrugs like FK506 or cyclosporine. Activators and inhibitors may beuseful for the therapeutical treatment of amyotrophic lateral sclerosis,Parkinson, Alzheimer, epilepsy, ischemia and cardiovascular diseases.

21. High Throughput BioAssay using recombinant calcineurin-A,recombinant calcineurin-B, calmodulin and recombinant CuZnSOD(analytical assay to identify activators or inhibitors ofCuZnSOD/calcineurin interaction)—Laser fluctuation correlationspectroscopy (FCS) is a useful tool to quantify ligand—ligandinteractions. The fluorescence F(t) of a optical well defined volumeelement which is excitated by a confocal laser is monitored as afunction of time. The temporal autocorrelation of the fluorescencefluctuation δ F(t) yields the time scale of this dynamics and theaverage number of independent fluorophores in the probe volume. If thefluorescene fluctuation arise from diffusive motion and fromfluorescence sensitive reaction, fluorescent fluctuation correlationfunction signal is approximated by the formula:G _(DR)(τ)=G _(Diff)(τ)*[1+A*exp(−k _(R)*τ)]

-   τ=fluorescence correlation time-   k_(R)=apparent binding constant of the fluorescent labeled ligand-   A=equilibrium coefficent dependent constant

If one measurement is performed with a solution only containing thefluorescent labeled ligand and a second measurement is performed with asolution containing the fluorescent labeled ligand and an interactingmolecule the correlation function G_(DR) can be separately analyzed andyields binding parameters of the interacting molecules. Upon binding ofthe ligand to the interacting molecule the hydrodynamic radius increasesand therefore the diffusion coefficient decreases resulting in a longercorrelation time.

A fluorescence labeled recombinant CuZnSOD as a fluorescent label wasused to monitor the binding dynamics to calcineurin. CuZnSOD was labeledwith Oregon-Green-514 dye according to the manufacturer's protocols(FluoReporter Protein labeling Kit, Molecular Probes, Leiden,Netherlands). The amount of fluorescent dye labels per CuZnSOD dimer wasquantified by determining the ratio of the absorbance at 265 nm (CuZnSODprotein)/514 nm (Oregon-dye). The diffusion constant and 110 correlationtime of the labeled CuZnSOD (100 nM) was measured on a bovine serumalbumin treated glass plate with an confocal laser microscope attachedto an autocorrelator (λ excitation=488 nm, λ emission=511 nm) in 10 μlassay buffer containing 50 mM sodiumphosphate pH 7.1, 150 mM NaCl, 0.67mM DTT, 0.67 μM calmodulin, 0.67 mM CaCl₂, 1 mM MgCl₂. The beam from amodelocked Ti:Sa or cw argon ionlaser was collimated to fill the backaperture of a immersion microscope objective (Zeiss C-Apochromat 63×1.2w), producing a small diffraction limited spot. The emitting fluorescentlight was collected by the same objective separated from the excitationlight by a beamsplitter/filter combination and imaged first to avariable pinhole and than to the detector (Avalanche Photodiode EG&GSPCM AQ161 or PMT Hamamatsu R5600-03). The labeled CuZnSOD exhibited anautocorrelation time corresponding to a hydrodynamic radius of 41,000Dalton which is comparable to the expected molecular weight of thehomodimer (34,600 Dalton). Next 0.2 μl calcineurin-A/B heterodimer (5μM) was added to a labeled CuZnSOD mixture and the fluorescencecorrelation signal was determined. The hydrodynamic radius increasesfrom 41 kDa to 90 kDa indicating that approximatly one calcineurinheterodimer interacts with one CuZnSOD dimer (expected: 114 kDa). Usingmutated D90A CuZnSOD yielded an apparent molecular weight of 180.000 kDaindicating the formation of calcineurin/CuZnSOD aggregates. The apparentbinding constant between human wild-type CuZnSOD and calcineurin wasestimated as kD=2×10⁻⁶ M+/−1×10⁻⁶ M. It is concluded that lasercorrelation spectroscopy is useful to perform a ultra high throughputscreening for ligands diminishing the CuZnSOD/calcineurin interactionwhich simply can be monitored by a reduction of the autocorrelation timeafter addition of a potential drugs. It is possible to screen forsuitable substances using substances available in chemical, peptide ornatural compound screening libraries.

22. High Throughput BioAssay using recombinant calcineurin-A,recombinant calcineurin-B, calmodulin and RII-Fluophos (analytical assayto identify activators or inhibitors of calcineurin)—RII peptide wassynthesized according to standard peptide synthesis protocols((Blumenthal et al., 1988); Interactiva, Ulm, Germany). To generate afluorescent labeled peptide which furthermore contains a phosphoester atSer-15, amino acid residue Ser-15 was coupled withfluorescein-phosphoamidit (FluoreDite Labeling Reagent, PerspectiveBiosystems), which is usually used for labeling of nucleotides, to yieldRII-Fluophos (Interactiva, Ulm, Germany). The expected molecular weight(2578.8 Dalton) was confirmed by mass spectrometry (2580.6 Dalton). TheFluophos-RII-peptide was not converted by calcineurin as was monitoredby fluorescence spectrometrie (Biolumin 960 UV-/VIS/fluorescencemicroplate reader). Therefore Fluophos-RII-peptide was used in laserfluorescence correlation spectroscopy as described in paragraph 20except that λ_(excitation) was 488 nm and λ_(emission) was 520 nm.Furthermore, labeled CuZnSOD was substituted by 10 nMFluophos-RII-peptide yielding a hydrodynamic radius corresponding to 4kDa (expected 2.6 kDa). After calcineurin addition the molecular weightincreases to 100.000 kDa and a binding constant of Kd=0.6×10⁻⁶ M isestimated. Binding constants were comparable between the six calcineurinisoforms/splice variants. It is concluded that laser correlationspectroscopy is useful to perform a ultra high throughput screening forligands directly substrate binding to calcineurin by simply monitoringthe autocorrelation time after addition of potential drugs. Bydescriminating the binding properties of potential drugs to the sixdifferent heterodimer combinations (calcineurin-A-alpha1/calcineurin-B,calcineurin-A-alpha2/calcineurin-B, calcineurin-A-beta1/calcineurin-B,calcineurin-A-beta2/calcineurin-B, calcineurin-A-gamma1/calcineurin-B,calcineurin-A-gamma2/calcineurin-B) it is possible to identify tissuespecific and therefore less toxic calcineurin inhibitors.

It is possible to combine the screening procedures described inparagraph 20 and 21 strategically: substances which are able to inhibitthe calcineurin-CuZnSOD interaction (positive hit in paragraph 20) butfailed to show an effect in procedure 21 (negative hit) arepredominantly positive candidates for the therapeutical use inneurological disorders because a toxic immunosuppressive side effect isless probable. Substances which fail to inhibit the calcineurin-CuZnSODinteraction (negative hit) but show an effect in procedure 21 (positivehit) are predominantly positive candidates for immunosuppression.Substances effective in both procedures are likely to be toxic.

23. Cellular BioAssay using eucaryotic cells transfected withcalcineurin-A-EGFP fusionprotein or CuZnSOD-EGFP fusion protein—PC12cells stably transfected with CuZnSOD and calcineurin isoenzymes serveas a model for monitoring the effects of CuZnSOD or calcineurinoverexpression in neuronal cells. CuZnSOD reportedly has been implicatedto be involved in the mediation of hypoxie tolerance, whereascalcineurin overexpression is associated with epileptogenesis,Parkinsonism or Alzheimers disease. It is possible to use theses cellssubsequently to the identification of potential drugs in screeningprotocols 20 und 21. Toxicity of potential neuroprotective drugs and theeffect on the subcellular distribution of calcineurin-isoforms orCuZnSOD, respectively can be monitored.

24. Pull-Down-BioAssay using histidine tagged recombinant CuZnSOD topurify CuZnSOD interacting ligands (Preparative assay to isolateactivators or inhibitors of CuZnSOD/calcineurin interaction frombiological sources)—Recombinant purified histidine tagged CuZnSOD in 50mM sodiumphosphate buffer pH 8.0 was attached to CuZn-NTA magneticagarose beads by incubating 100 μl beads suspension with 100 μl CuZnSODsolution (0.3 μg/μl) in 96 well microplates for 30 minutes at roomtemperature on a microplate shaker (600 rpm). CuZn-NTA magnetic beadswere generated from Ni-NTA beads by applying the same procedure asdescribed under paragraph 5 (Ni-NTA magnetic agarose beads, Qiagen,Hilden, Germany). The microplate was placed on the 96 well magnet for 1minute and the supernatant removed from the wells.

Cytosolic ligands were isolated as follows: 200 μl interaction buffer(50 mM NaH₂PO₄, 300 mM NaCl, 20 mM imidazole pH 8.0, 0.1% Tween-80) wereadded to the CuZn-NTA agarose beads/CuZnSOD containig wells and placedon the 96 well magnet to remove interaction buffer. 100 mg tissue, cellsor other biological specimen to be analyzed for CuZnSOD interactingligands were homogenized in 200 μl lysis buffer (50 mM NaH₂PO₄, 300 mMNaCl, 10 mM imidazole pH 8.0, 0.1% Tween-80) using a dounce homogenizer.The lysate was cleared by 30 min centrifugation at 10,000 g at 4° C. Thesupernatant was applied to the wells containing CuZn-NTA absorbedrecombinant human CuZnSOD, mixed and incubated for 60 minutes at 0° C.The microplate was placed on the 96 well magnet for 1 minute to removethe supernatant. After removal of the lysate the wells were washed twiceby adding 200 μl interaction buffer. Elution of CuZnSOD and interactingligands was achieved by addition of 100 μl elution buffer (50 mMNaH₂PO₄, 300 mM NaCl, 250 mM imidazole pH 8.0, 0.1% Tween-80).

Membranous ligands were isolated as follows: denaturing interactionbuffer (6 M guanidine-HCL, 100 mM NaH₂PO₄ pH 8.0, 0.1% Tween-80) wasadded to the CuZn-NTA agarose beads/CuZnSOD containig wells and placedon the 96 well magnet to remove interaction buffer. The pellet from theprocedure above was solubilized in 200 μl denaturing interaction buffer(6 M guanidine-HCL, 100 mM NaH₂PO₄ pH 8.0, 0.1% Tween-80) for 60 minutesat room temperature. The solubilisate was cleared by 30 mincentrifugation at 10,000 g at room temperature. The supernatant wasapplied to the wells containing CuZn-NTA absorbed-recombinant humanCuZnSOD, mixed and incubated for 60 minutes at room temperature. Themicroplate was placed on the 96 well magnet for 1 minute to remove thesupernatant. The wells were washed once with 200 μl denaturinginteraction buffer (6 M guanidine-HCL, 100 mM NaH₂PO₄ pH 8.0, 0.1%Tween-80) and a second time with 200 μl denaturing wash buffer (8 MUrea, 100 mM NaH2PO4 pH 8.0, 0.1% Tween-80). Elution of CuZnSOD andinteracting ligands was achieved by addition of 100 μl denaturingelution buffer (8 M Urea, 100 mM NaH₂PO₄ pH 4.0, 0.1% Tween-80).

To remove low molecular weight ligands for HPLC analysis, the eluates(cytosolic or membranous) were ultrafiltered through a 5 kDa membrane asdescribed under 7. Low molecular weight ligands were separated on apreparative reverse phase HPLC (UV detection at 200 nm). Homogeneity andmolecular weight of UV detectable fractions were analyzed by massspectrometry. High molecular weight ligands (ultrafiltration remainder)were separated on a 10% polyacrylamide gel and protein bands identifiedby sequencing or MALDI mass spectrometrie as described under 19.Interacting nucleic acid was analyzed by separating the membranouseluate on a 1% agarose gel and staining with ethidium bromide.Fluorescent bands were extracted from the agarose (Qiagen gel extraktionkit, Qiagen, Hilden, Germany) subjected to digestion with RsaI andsubcloned into RsaI treated pQE30 vector for DNA sequencing.

25. Pull-Down-BioAssay using histidine tagged recombinant calcineurin-Aand calcineurin-B to purify calcineurin interacting ligands (Preparativeassay to isolate activators or inhibitors of CuZnSOD/calcineurininteraction from biological sources)—Isolation and identification ofcalcineurin interacting ligands was performed analogous to paragraph 24with the exception that recombinant calcineurin-A/B heterodimer wasattached to Fe-NTA magnetic agarose beads which were prepared asdescribed under paragraph 15. Furthermore six different heterodimercombinations were used (calcineurin-A-alpha1/calcineurin-B,calcineurin-A-alpha2/calcineurin-B, calcineurin-A-beta1/calcineurin-B,calcineurin-A-beta2/calcineurin-B, calcineurin-A-gamma1/calcineurin-B,calcineurin-A-gamma2/calcineurin-B) to descriminate between isoenzymeand splice variant specific interaction partners.

LITERATURE

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Content of Sequence Listing

-   1. eukaryotic expression vector CuZnSOD-EGFP (CuZnSOD-pEGFP) (DNA)-   2. CuZnSOD (PRT)-   3. EGFP (PRT)-   4. eukaryotic expression vector EGFP-Calcineurin A alpha    (CNAa-pEGFP) (DNA)-   5. EGFP (PRT)-   6. Calcineurin A alpha (PRT)-   7. eukaryotic expression vector EGFP-Calcineurin A beta (CNAb-pEGFP)    (DNA)-   8. EGFP (PRT)-   9. Calcineurin A beta (PRT)-   10. eukaryotic expression vector EGFP-Calcineurin A gamma    (CNAg-pEGFP) (DNA)-   11. EGFP (PRT)-   12. Calcineurin A gamma (PRT)-   13. prokaryotic expression vector His-CuZnSOD (CuZnSOD-pQE30) (DNA)-   14. CuZnSOD (PRT)-   15. prokaryotic expression vector CuZnSOD-His (CuZnSOD-pQE60) (DNA)-   16. CuZnSOD (PRT)-   17. prokaryotic expression vector His-Calcineurin A    alpha1-Calcineurin B (CNAa1-pQE30) (DNA)-   18. Calcineurin A alpha1 (PRT)-   19. Calcineurin B (PRT)-   20. prokaryotic expression vector His-Calcineurin A    alpha2-Calcineurin B (CNAa2-pQE30) (DNA)-   21. Calcineurin A alpha2 (PRT)-   22. Calcineurin B (PRT)-   23. prokaryotic expression vector His-Calcineurin A    beta1-Calcineurin B (CNAb1-pQE30) (DNA)-   24. Calcineurin A beta1 (PRT)-   25. Calcineurin B (PRT)-   26. prokaryotic expression vector His-Calcineurin A    beta2-Calcineurin B (CNAb2-pQE30) (DNA)-   27. Calcineurin A beta2 (PRT)-   28. Calcineurin B (PRT)-   29. prokaryotic expression vector His-Calcineurin A    gamma1-Calcineurin B (CNAg1-pQE30) (DNA)-   30. Calcineurin A gamma1 (PRT)-   31. Calcineurin B (PRT)-   32. prokaryotic expression vector His-Calcineurin A    gamma2-Calcineurin B (CNAg2-pQE30) (DNA)-   33. Calcineurin A gamma2 (PRT)-   34. Calcineurin B (PRT)-   35. peptide RII (PRT)

1. A method for screening for a modulator of calcineurin enzymaticactivity, characterized in that a direct interaction between mammaliancalcineurin and mammalian Cu/Zn-superoxide dismutase is monitored,comprising the following steps formation of a complex comprising atleast mammalian calcineurin and mammalian Cu/Zn-superoxide dismutase inthe presence of at least one potential modulator, detecting an influenceof the potential modulator by directly monitoring the complex formation.2. The method according to claim 1, characterized in that the monitoringis performed by detection of labels.
 3. The method according to claim 1,characterized in that the mammalian calcineurin and/or the mammalianCu/Zn-superoxide dismutase carry labels, wherein the labels are enhancedgreen fluorescent protein.
 4. The method according to claim 3,characterized in that mammalian calcineurin and/or mammalianCu/Zn-superoxide dismutase are expressed as fluorescent proteins.
 5. Themethod according to claim 1, characterized in that the monitoring ofcomplex formation is performed by laser fluctuation correlationspectroscopy.
 6. The method according to claim 1, characterized in thatmammalian calcineurin and mammalian Cu/Zn-superoxide dismutase arecoexpressed in cells, and that the complex formation is performed withinthe cell.
 7. The method according to claim 1, characterized in thatmammalian calcineurin and/or mammalian Cu/Zn-superoxide dismutase areexpressed in cells, and that mammalian calcineurin and/or mammalianCu/Zn-superoxide dismutase are isolated and/or purified before thecomplex formation is performed.
 8. The method according to claim 7,characterized in that purification of mammalian calcineurin is achievedby ferro-nitrilotriacetat(NTA)-metal affinity chromatography.
 9. Themethod according to claim 7, characterized in that purification ofmammalian Cu/Zn-superoxide dismutase is achieved bycopper/zinc-NTA-metal affinity chromatography.
 10. The method accordingto claim 1, characterized in that in the complex formation step,calmodulin and/or calcium are present.
 11. The method according to claim1, characterized in that additionally a monitoring of the enzymaticactivity is performed by analyzing the phosphatase activity of mammaliancalcineurin.
 12. The method according to claim 11, characterized in thatthe phosphatase activity is analyzed by the use of at least onesubstrate, which carries a label.
 13. The method according to claim 12,characterized in that the substrate is a peptide characterized by theamino acid sequenceAsp-Leu-Asp-Val-Pro-Ile-Pro-Gly-Arg-Phe-Asp-Arg-Arg-Val-Ser-Val-Ala-Ala-Glu.14. The method according to claim 12, characterized in that thesubstrate is a peptide containing a residue labeled with fluoresceine.15. The method according to claim 11, characterized in that theinfluence of the potential modulator on the enzymatic activity isdetected separately from the influence of the potential modulator on thecomplex formation.
 16. A method for screening of modulators of mammaliancalcineurin activity, comprising: a) determining the interaction of apotential modulator with either mammalian calcineurin or mammalianCu/Zn-superoxide dismutase as a partner, b) taking a potential modulatorshowing interaction with mammalian calcineurin or mammalianCu/Zn-superoxide dismutase according to step a), c) determining theinteraction of said modulator taken in step b), with the other partner,namely mammalian calcineurin or mammalian Cu/Zn-superoxide dismutase,respectively, and d) identifying the potential modulator showinginteraction also according to step c).
 17. The method according to claim16, characterized in that mammalian calcineurin and/or mammalianCu/Zn-superoxide dismutase comprises at least one tag.
 18. The methodaccording to claim 16, characterized in that mammalian calcineurinand/or mammalian Cu/Zn-superoxide dismutase is attached to a solidmatrix.
 19. The method according to claim 1, characterized in that saidmammalian calcineurin is human calcineurin.
 20. The method according toclaim 19, characterized in that said human calcineurin is a combinationof a calcineurin A subunit selected from the group consisting of A-α1,A-α2, A-β1, A-β2, A-γ1, and A-γ2, and calcineurin B.
 21. The methodaccording to claim 16, characterized in that said mammalian calcineurinis human calcineurin.
 22. The method according to claim 21,characterized in that said human calcineurin is a combination of acalcineurin A subunit selected from the group consisting of A-α1, A-α2,A-β1, A-β52, A-γ1, and A-γ2, and calcineurin B.